Epithelial chloride channel 3

Question 1

some papers on discovery of CF gene product

Answer 1

1989
Rommens, Riordan and Kerem
identifying CF gene

Question 2

CF gene

Answer 2

chromosome 7
250, 000 base pairs
gene product = CFTR
deltaF508 mutation >70%

Question 3

CFTR has structural homology with what?

Answer 3

ATP binding cassette ABC

superfamily of transport proteins

Question 4

3 domains of CFTR

Answer 4

2 hydrophobic membrane spanning domains
2 nucleotide binding domains
unique regulatory domain

Question 5

MSDs of CFTR

Answer 5

each comprised of 6 alpha helices

Question 6

NBDs od CFTR

Answer 6

bind and hydrolyse ATP

NBD1 - site of deltaF508mutation

Question 7

Unique R domain of CFTR

Answer 7

phosphorylation site for PKA

Question 8

Rich et al 1990

Answer 8

CFTR expression correct defective Cl channel regulation in CF airway

Question 9

1990 - Rich et al explain

Answer 9

transfect CF cells CFTR
normal cAMP activated Cl currents
deltaF508 did not do this
channels induced by CFTR expression similar to SLCCs

Question 10

Speculation by Rich et al in 1990

Answer 10

CFTR is a SLCC

Question 11

S9 insect cell line CFTR

Answer 11

no chloride conductance
can you know that?
mRNA - CFTR to membrane purified CFTR
protein mixed with phospholipids to make vesicles
CFTR into vesicles
plate with 200microns hole
drop vesicle into chamber
CFTR incorporated into synthetic membrane

Question 12

1992 Bear

Answer 12

purification and functional reconstitution of CFTR

purified CFTR incorporated into planar lipid bilayers

Question 13

1992 bear - what was found?

Answer 13

cAMP activated SLCC

Question 14

what type of channel is CFTR?

Answer 14

low conductance, cAMP activated chloride channel

SLCC

Question 15

Kartner 1992

Answer 15

deltaF508 CFTR in CF sweat gland

Question 16

kartner 1992 - what happened to deltaF508 mutated CFTR?

Answer 16

did not localise to apical membrane

CF may be due to absence of Cl channels rather than defective regulation

Question 17

What did Denning confirm in 1992?

Answer 17

Kartner et al 1992

Question 18

Denning 1992 - temperature

Answer 18

lowering temperature permits dF508 to localise to apical membrane?

Question 19

Sheppard 1993

Answer 19

Mutations in CFTR mild-disease form Cl channels with altered pore properties

Question 20

Sheppard 1993 findings

Answer 20

3 mutations in 1st MSD of CFTR
affects arginine residues
apical cAMP activated SLCC with reduced currents

Question 21

what did sheppard in 1993 find that might determine Cl conductivity of channel?

Answer 21

arginine residues

Question 22

Cotten and Welsh 1999

Answer 22

arginine 347 mutations alter pore architecture of CFTR

Question 23

findings from Cotten and Welsh 1999

Answer 23

Electrostatic interaction between actionic and anionic amino acid residues stabilises channel structure
asp –> arg or vice versa destabilise channel
switch both simultaneously = no problem

Question 24

Welsh and Smith 1993 - 4 main potential problems of CFTR

Answer 24

protein production
processing - dF508 not get from ER to membrane
regulation - protein in membrane but not activated by cAMP, PKA
conduction - chloride